Monoclonal antibodies to human leukocyte antigens

ABSTRACT

Monoclonal antibodies, Ta60b(Ferm BP-2170) and Ts145(Ferm BP-2171), are used for detecting human leukocyte antigens such as T cell differentiation antigens and T cell subset antigens.

TECHNICAL FIELD

The present invention relates to monoclonal antibodies for detecting anantigen present in human leukocytes, and to a process for producing thesame.

BACKGROUND ART

It has become possible to produce monoclonal antibodies to T cells, Bcells and their individual subsets of human lymphocytes by recentlydeveloped cell technological procedures, and the individual cell groupshave been gradually clarified [Reinherz, E. L. et al: Cell 19, 821(1980), Foon, K. A. et al: Blood 60, 1 (1982)].

That is, functional cell subsets of human lymphocytes have been muchclarified by using monoclonal antibodies recognizing these subsets, andtheir abnormalities in various diseases have been gradually clarified.

DISCLOSURE OF INVENTION

As a result of extensive studies to find monoclonal antibodiesapplicable to diagnosis of various diseases of haemopoietic organs,etc., the present inventors have found monoclonal antibodies to theantigens present in leukocytes and to adult T cell leukemia (hereinafterreferred to as "ATL"), and have established the present invention.

The present invention is explained in detail below:

The present invention provides monoclonal antibodies to the antigenspresent in leukocytes and ATL virus (hereinafter referred to as "ATLV").

Monoclonal antibodies of the present invention include those calledTpw40, Tp120, Ta60a, Ta60b, Ta60c, Ts60, Tsw32, TsA, TsB, Ts145, Lp95,Ls70, LsA, ATV19a, and ATV19b.

Ta60a, Ta60b and Ta60c characteristically fail to react with normalblood cells such as normal human peripheral blood T cell fraction,non-T(B) cell fraction, monocytes, granulocytes, thymocytes, etc but canreact with the so-called activated T cells obtained by stimulating Tcell groups by mitogens such as interleukin-2 [IL-2, Biotest Co. (WestGermany)], phytohaemaglutinin [PHA, Difco Co. (USA)], concanavalin A[ConA, E.T Co. (USA)], pokeweed mitogen [PWM, Gibco Co. (USA)], etc.,allo B cells, and the like (see Example 8). That is, they arecharacterized in that no response is observed under culturing conditionsonly with a culture liquor containing no mitogen as a control

Among the monoclonal antibodies of the present invention, Ta60a andTa60b are positive to all the cases of ATL among those derived from Tcells in the haemopoietic tumor cells failing to react with the cellsderived from solid tumors, and are negative to substantially all thecases of malignant diseases derived from T cells other than ATL. Thisshows that the monoclonal antibodies of the present invention can beused for clearly distinguishing clinically hard-to-discriminate casesfrom one another

As for the tumor cells other than those derived from T cells, themonoclonal antibodies of the present invention characteristicallyundergo weakly positive reaction with some cases of B-CLL (B cell-typechronic lymphatic leukemia) and B-ML (B cell-type malignant lymphoma)and also undergo weakly positive reaction with some cases of AML (acutemyeloid leukemia) derived from myelocytes

Tpw40 and Tpl20 react with most cells of T cell fraction of peripheralblood lymphocytes and detect the so-called T cells (panT).

Five antibodies of Ts60, Tsw32, TsA, TsB and Ts145 detect antigenspresent in some cells of T cell fraction and T subset antigens Ts60 hasthe same antigen molecular weight as that of Ta60, but has a differentserological specificity. It does not react with activated T cells, butonly with some of T cell strains.

Lp95 detects pan leukocyte antigen reacting with all the leukocytes,whereas Ls70 and LsA react with monocytes and some of other leukocytesand recognize leukocyte subset antigens.

Both Tpw40 and Tpl20 detect panT antigens Tpw40 reacts with relativelyimmature T cell-type acute lymphatic leukemia (T-ALL) and lymphoblast(LL) in the T cell tumor, whereas Tpl20 reacts with T₂ lymphoma and ATLwhich are mature T cell tumors Furthermore, Tpl20 reacts with Bcell-type chronic lymphatic leukemia (B-CLL) (Table 6).

Tsw32 reacts with T-ALL and LL, and TsA reacts with both these T celltumors and also with mature T cell tumors such as T₂ lymphoma, ATL, etc.

TsB reacts with T₂ lymphoma and ATL which are mature T cell tumors to ahigh degree. TsB also reacts with B-CLL.

These antibodies capable of detecting T cell-differentiating antigensare useful for discriminating and diagnosing T cell tumors

Ts145 hardly reacts with peripheral lymphocytes, but characteristicallyundergoes reaction specifically with T cell strains cultured for a longterm in the presence of IL-2.

Lp95 reacts with most leukocytes and also reacts with substantially allof leukemia and lymphoma.

Ls70 exists in monocytes and some of other leukocytes and reacts with T₂lymphoma and ATL among T cell lymphomas and also reacts with B-CLL.

LsA reacts mainly with monocytes and T cells, and never reacts with Tcell lymphoma, but reacts with monocytic leukemia to a high degree.

ATV19a and ATV19b antibodies are monoclonal antibodies to P19 componentin ATLV-constituting protein, and characteristically detect antigenspresent in ATL-derived culture strains such as MT-1, MT-2, etc. and inATL leukemia cells cultured for a short term in the presence of IL-2.

ATV19a and ATV19b recognize peptides having a molecular weight of 19×10³which are virus particle-constituting protein, and are useful fordiagnosis of ATL.

Biological activities of the monoclonal antibodies of the presentinvention are shown in Examples 6-13. Mouse-mixed hemadsorption assay,M-MHA used in Examples is carried out according to the following methodwherein observation is made of presence or absence of the attachment ofmarker cells to target cells prepared by depositing a single layer cellculture or suspended cells onto a microplate (3040, Falcon Co.). Themarker cell is prepared by reacting sheep erythrocyte with mouseanti-sheep erythrocyte antibody, followed by reaction with rabbitanti-mouse immunogloblin serum.

Mouse-mixed hemadsorption assay [M-MHA]:

M-MHA is carried out by a modification of the Espmark and Fagreus method(Acta Pathol Microbiol. Second Suppl. 154, 258-262, 1962)

The method for preparing marker cells is as follows.

A 2% suspension of washed sheep erythrocytes is reacted with an equalvolume of 1,000-fold dilution of mouse anti-sheep erythrocyte antibody(prepared by overimmunizing sheep erythrocytes on BALB/c mouse) at 24°C. for 45 minutes, and then washed. The resulting sheep erythrocytes arereacted, as a 2% suspension, with an equal volume of 200-fold dilutionof rabbit anti-mouse immunoglobulin (Cappel Co., USA) at 24° C. for 45minutes, and then washed twice. A 2% suspension of the resulting sheeperythrocytes is used as the marker erythrocyte for M-MHA

A technique for M-MHA comprises the steps of reacting cells to bedetected with hybridoma cells, culture supernatant, or ascites ofhybridoma cell-inoculated BALB/c mouse or BALB/c-derived nude mouse at24° C. for 45 minutes; removing antibodies therefrom by washing;reacting the cells with 0.2% marker sheep erythrocytes at 24° C. for 45minutes; lightly washing the cells with a phosphate buffer-physiologicalsaline solution (PBS); and then detecting the presence or absence ofrosette formation of the marker erythrocytes with an optical microscope

Indirect fluorescent antibody method [cytoplasm fluorescent antibodymethod (Cy-IF) or membrane fluorescent antibody method (M-IF)] wascarried out in the following manner.

Cytoplasm fluorescent antibody method

As marker cells, 1-5×10⁴ target cells fixed on a slide glass with 95%acetone for 10 minutes and reserved at -70° C. are used.

As a primary antibody, 25 μl of hybridoma cell culture supernatant orascites of hybridoma cell-inoculated BALB/c mouse or BALB/c-derived nudemouse is used for reaction at 4° C. or 25° C. for 30 minutes. Afterwashing with PBS, 20-40-fold diluted fluorsscence labelled rabbitmouse-immunogloblin GAM (GAM-FITC, Coulter Corp , USA) as a secondaryantibody is used for reaction at 25° C. for 30 minutes. The result ofthe reaction is judged by observing the presence or absence offluorescence with a fluorescence microscope.

Membrane fluorescent antibody method 50 μl of 5×10⁵ target cells/ml and50 μl of hybridoma cell culture supernatant or ascites of hybridomacell-inoculated BALB/c mouse or BALB/c-derived nude mouse are used forreaction at 4° C. or 25° C. for 30 minutes. After washing with PBS,20-40-fold diluted fluorescence labelled rabbit mouse-immunogloblin GAM(GAM-FITC, Coulter Corp., USA) as a secondary antibody is used forreaction at 25° C. for 30 minutes. The result of the reaction is judgedby observing the presence or absence of fluorescence with afluorescence--microscope.

The monoclonal antibodies of the present invention can be prepared byimmunizing mice with ATL cells, ATLV-productive strain MT-2 cells, ATLvirus (ATLV) separated from MT-2, mixed cultured lymphocytes, protein A(PA) activated lymphocytes, etc., sampling spleen cells therefrom, andculturing hybridoma obtained by fusing the spleen cells with mousemyeloma cells.

The preparation of the hybridoma can be carried out according to themethod of Ueda, et al [Proc Natl. Acad. Sci. USA 78, 5122-5126 (1981)],which is a modification of the method of Kohler and Milstein [Nature256, 495-497 (1975)].

As mice for immunization, BALB/c mice, Fl mice derived from BALB/c miceand mice of other species, etc. can be used.

Immunization is carried out using 10⁷⁻⁸ cells or 50-500 μg of virus fora mouse (age of 4-8 weeks, 20-30 g) twice or three times at an intervalof 2-5 weeks. Breeding of mice and sampling of spleen cells are carriedout in the ordinary manner.

As myeloma cells, MOPC-21 NS/l [Nature, 256, 495-497 (1975)], SP2/0-Ag14[Nature 277, 131-133 (1979)], S194/5, XXO. BU. 1 [J. Exp. Med. 148,313-328 (1978)], etc. can be used. Spleen cells and myeloma cells aremixed together in a ratio of 1:1-10:1, and fusion is carried out in aphosphate buffer solution (pH 7.2-7.4) oontaining about 0.85% NaCl,10-20% (V/V) dimethyl sulfoxide and polyethylene glycol having amolecular weight of 1,000-6,000.

Fusion is carried out by incubating both cells at 35°-37° C. for 1-3minutes

Selection of fused cells (hybridoma) is carried out by selecting thecells which grow in a basal medium containing 1.3-1.4 mg/dlhypoxanthine, 18-20 μg/dl aminopterin, 375-400 μg/dl thymidine, 50-100μg/ml streptomycin, 50-100 units of penicillin, 3.5-4.0 g/l glutamine,and 10-20% fetal calf serum.

As the basal medium, RPMI1640 medium, Eagle's MEM medium, etc. which aregenerally used for culturing animal cells can be used.

Cloning of fused cells (hybridoma) must be repeated at least three timesaccording to a limiting dilution procedure.

By culturing the hybridoma in the same manner as in the ordinaryculturing of animal cells, the antibody of the present invention can beproduced in a medium For example, when 2-5×10⁶ hybridoma cells arecultured in a flask containing 10-20 ml of RPMI1640 medium containing50-100 μg/ml streptomycin, 50-100 units/ml penicillin, 3.5-4.0 g/lglutamin and 10-20% fetal calf serum in the presence of 95% CO₂ -5% O₂at 35°-37° C. for 3-7 days, the antibody is secreted and accumulated inthe culture medium.

When the hybridoma cells are transplanted into the abdominal cavity ofPristane-treated nude mouse or BALB/c mouse, and propagated, theantibody of the present invention is accumulated in the ascites. In thiscase, 0.5-1 ml of Pristane (2,6,10,14-tetramethylpentadecane, made byAldrich Corp., USA) is injected into abdominal cavities of these mice,and 2-3 weeks thereafter, 5-10×10⁶ hybridoma cells are transplantedtherein Usually, 7-10 days thereafter, ascites starts to accumulate, andis sampled.

In the present invention, ATL-derived cell strain MT-2 is used as animmunogen to obtain monoclonal antibodies Ta60b, Ta60c and Ts60, andATLV is used as an immunogen to obtain monoclonal antibodies Ta60a,ATV19a and ATV19b.

ATL cells are used as an immunogen to obtain TsA, mixed lymphocytecultured cells are used as an immunogen to obtain Tpw40, Tpl20, Tsw32,Lp95, Ls70 and LsA, and PA-activated lymphocyte is used as an immunogento obtain TsB and Ts145.

Antigen specificity of these monoclonal antibodies has been confirmed inthe manners shown in Examples 6-13.

As for the antibody class of the monoclonal antibodies of the presentinvention, Tpl20 and Lp95 are classed as IgG2a, TsA as IgM, and all theothers as IgGl. According to the molecular weight determination usingthe following immunological precipitation reaction by [³ H]-glucosaminelabelling, Ta60a, Ta60b, Ta60c and Ts60 antigens showed 60,000 daltons(gp60 MT-2) in MT-2 or MT-1 cells and 53,000 daltons (gp53 HUT102) inHUT102 cells, and these 4 antigents showed substantially equal molecularweights.

Tpl20 antigen showed 120,000 daltons in HUT120 cells.

According to the molecular weight determination using immunologicalprecipitation reaction by ¹²⁵ I labelling, Ts145 antigen and Ls70antigen showed 145,000 daltons and 70,000 daltons, respectively, inNK3.3 cells.

According to the molecular weight determination by ³⁵ S methioninelabelling, Lp95 antigen showed 95,000 daltons in HUT102 cells.

According to the molecular weight determination by immunoblottingmethod, both ATV19a and ATV19b showed the same molecular weight as thatof the virus particle constituent of ATLV having 19,000 daltons.

Molecular weights of the other five antigens (Tpw40, Tsw32, TsA, TsB andLsA) have not been clear yet.

Immunological precipitation reaction

Target cells (MT-2, MT-1 and HUT102) are labelled with [³H]-glucosamine. Labelling is carried out by culturing the cells inRPMI1640 culture medium (made by Nissui Seiyaku Co.) containing 15μCi/ml ³ H-glucosamine (38 Curies/mmol, Amersham, USA) for 35-40 hours.

Target cells HUT102 are labelled with ³⁵ S methionine Labelling wascarried out by culturing the cells in RPMI1640 culture medium containing500 μCi/ml ³⁵ S methionine for 8 hours. 5×10⁷ NK3.3 target cells arelabelled with 0.5 mCi of Na¹²⁵ I in the presence of 200 μg of iodogen.

These labelled cells are solubilized with 0.1-1% (V/V) NP-40, and theextract of the cells (1-10×10⁵ cpm) is reacted with 1-10 μl ofmonoclonal antibodies at 4° C. for 6-12 hours. Then, the cells arereacted with 5-20 μl of rabbit anti-mouse immunogloblin (Cappel Corp ,USA) as a secondary antibody at 4° C. for 30 minutes, and furtherreacted with Staphylococcus aureus (Cowan I strain) at 4° C. for onehour to prepare immunological precipitates, and the precipitates areanalyzed by SDS-polyacrylamide gel electrophoresis.

Immunoblotting method (Towin, H. et al: Proc. Natl. Acad. Sci. USA 76,4350, 1979):

Development is made according to polyacrylamide gel electrophoresis, andthe individual peptides are transferred onto a nitrocellulose membrane.The peptides are reacted with a monoclonal antibody, and further reactedwith ¹³¹ I-labelled anti-mouse immunogloblin antibody as a secondaryantibody, and then observed by fluorography.

The monoclonal antibodies of the present invention are disclosed in thearticles of the 42nd general meeting of Japan Cancer Society, page 100,No. 290 and Symposium VI23, No. 23 published by Japan Cancer Society onSept. 25, 1983, wherein TA-53c, TA-53b, TA-53a, TS-53, ATV-W26a andATV-W26b correspond to Ta60a, Ta60b, Ta60c, Ts60, ATV19a and ATV19b,respectively, in the present specification. TP-W40, LP-94, TP-W67 andTP-120 disclosed in Summary of Japan Immunological Society, published onNov. 10, 1983, correspond to Tpw40, Lp95, TsA and Tpl20, respectively,in the present specification.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1 Preparation ofmonoclonal antibodies Ta60a, Ta60c, ATV19a and ATV19b:

Virus particle protein fractions separated from culture supernatant ofATL-derived cultured strain MT-2 [Gann 71, 155 (1980)]were used as animmunogen [Yoshida, H. et al: Primary and tertiary structure of nucleicacids and cancer research, M. Miwa et al (EDS, Japan. Sci. Soc. Press,Tokyo, pp. 255-294, 1982)].

Said protein fractions (protein amount: 0.45 mg) were subjected to 6repetitions of a freezing-thawing process consisting of freezinginstantly in ethyl alcohol containing dry ice and thawing in athermostat tank at 37° C., just before immunization, and then mixed with0.02 ml of complete Freund's adjuvant and subcutaneously inoculated ontothe back of a BALB/c mouse (age of 8 weeks, 24 g) (purchased fromCharles River, Inc., Atsugi, Japan) for immunization. The immunizationwas carried out four times at an interval of 4 weeks, and the mouse waskilled on the 4th day after the final immunization Spleen was excised,and suspended cells were obtained in the ordinary manner.

Then, 10⁸ suspended cells of the spleen and 2×10⁷ mouse myelomaMOPC-21NS/l cells were fused in 0.2 ml of phosphate buffer-physiologicalsaline solution (PBS) containing 42% (W/V) polyethylene glycol (M.W.4,000, Koch-Light, Bucks, Great Britain) and 15% (V/V) dimethylsulfoxide (Merck, Darmstadt, West Germany) according to the method ofUeda et al [Proc Natl. Acad. Sci., USA 78, 5122-5126 (1981)].

Fused cells were selected in HAT medium [RPMI1640 medium (pH 7.2)containing 1.36 mg/dl hypoxanthine, 19.1 μg/dl aminopterin, 387 μg/dlthymidine, 100 μg/ml streptomycin, 100 units/ml penicillin, 2 mMglutamine and 10% fetal calf serum] according to the ordinary method(said method of Ueda et al).

Three fused cells thus obtained were subjected three times to thelimiting dilution method to make cloning. The obtained three clones werecultured in the following manner to obtain strains capable of producinga remarkable amount of monoclonal antibodies which specifically reactwith ATL-related antigens. That is, 5×10⁶ hybridoma cells were culturedin a 150 cm³ -flask containing 15 ml of RPMI1640 medium containing 100μg/ml streptomycin, 100 units/ml penicillin, 3.8 g/l glutamine and 10%fetal calf serum in the presence of 95% CO₂ - 5% 02 at 37° C. for 7days.

Monoclonal antibodies produced by the thus obtained cell strains Ta60a,Ta60c, ATV19a and ATV19b were designated as Ta60a, Ta60c, ATV19a andATV19b, respectively

The cell strains were cultured by administering them into the abdominalcavity of a mouse according to the following method, whereby 3-10 mg/leach of Ta60a, Ta60c, ATV19a and ATV19b were formed. That is, 0.5 ml ofPristane was injected into the abdominal cavity of a mouse, and threeweeks thereafter, 5×10⁶ hybridoma cells were transplanted into theabdominal cavity. Ten days thereafter, ascites was sampled.

EXAMPLE 2 Preparation of monoclonal antibodies Ta60b and Ts60

A mouse was initially immunized subcutaneously with 10⁷ ATL-derivedcultured strains MT-2 [Miyoshi et al: Gann 71, 155 (1980)]and subjectedto the second immunization in the same manner as above three weeksthereafter. Then, the third immunization was carried out by injection of10⁷ MT-2 strains into the abdominal cavity. On the 4th day thereafter,the immuned mouse was killed, and spleen cells were sampled andsubjected to cell fusion. Then, monoclonal antibodies Ta60b and Ts60were obtained in the same manner as in Example 1.

EXAMPLE 3 Preparation of monoclonal antibody TsA:

A mouse was initially immunized subcutaneously with 10⁷ ATL cellsseparated from the peripheral blood of an ATL patient, and subjected tothe second immunization in the same manner as above three weeksthereafter. Furthermore, the third immunization was carried out byinjection of 10⁷ ATL cells into the abdominal cavity. On the 4th daythereafter, the immuned mouse was killed, and spleen cells were sampledand subjected to cell fusion Then, monoclonal antibody TsA was obtainedin the same manner as in Example 1.

EXAMPLE 4 Preparation of monoclonal antibodies Tpw40, Tpl20, Tsw32,Lp95, Ls70 and LsA:

Lymphocytes were sampled on the 4th day of mixed culturing and washed,and a mouse was initially immunized subcutaneously with 5×10⁶ cells thusobtained, and subjected to the second immunization three weeksthereafter in the same manner as above. The third immunization wascarried out by injection of 10⁷ cells into the abdominal cavity. On the4th day thereafter, the immuned mouse was killed, and spleen cells weresampled and subjected to cell fusion. Then, the above-mentionedmonoclonal antibodies were obtained in the same manner as in Example 1.

EXAMPLE 5 Preparation of monoclonal antibodies TsB and Ts145

Peripheral blood lymphocytes were cultured in the presence of 10 μg/mlprotein A (PA) for 3 days, and activated lymphocytes were sampled. Afterwashing of the cells, a mouse was initially immunized subcutaneouslywith 5×10⁶ cells thus obtained, and subjected to the second immunizationthree weeks thereafter in the same manner as above. Then, the thirdimmunization was carried out by injection of 10⁷ cells into theabdominal cavity. On the 4th day thereafter, the immuned mouse waskilled, and spleen cells were sampled and subjected to cell fusion Then,monoclonal antibodies TsB and Ts145 were obtained in the same manner asin Example 1.

EXAMPLE 6 Reactivity of monoclonal antibodies Ta60a, Ta60b, Ts60 andATV19a to cells of various cultured strains and human normal blood cellcomponents:

To investigate the reaction of cell surface antigens with theabove-mentioned monoclonal antibodies, the mouse-mixed hemadsorptionassay [M-MHA] described above was carried out. The reaction ofintraplasm antigens with the antibodies was investigated by the indirectfluorescent antibody method described above The results are shown inTable 1.

                                      TABLE 1                                     __________________________________________________________________________                    Cell surface antigen                                                                           Intraplasm antigen                                           (M-MHA inspection)                                                                             (Cy-IF inspection#)                                          Ta60a                                                                             Ta60b                                                                             Ts60                                                                              ATV19a                                                                             Ta60a                                                                              Ta60b                                                                             Ts60                                                                              ATV19a                          __________________________________________________________________________    ATL related                                                                   MT-2            +++ +++ +++ -    +++  +++ +++ +++                             MT-1            +++ +++ -   -    +++* +++ ±                                                                              +++ *                           HUT102          +++ +++ +++ -    +++  +++ +++ +++                             HUT78           -   -   +++ -    -    -   +++ -                               ATN-1           +++ +++ -   -    +++  +++ ±                                                                              +++                             T cell                                                                        RPMI8402        -   -   -   -    -    -   -   -                               HPB-ALL         -   -   -   -    -    -   -   -                               MOLT-3          -   -   -   -    -    -   -   -                               Jurkat          -   -   +++ -    -    -   -   -                               IL-2 dependent                                                                NK3.3           +++ +++ -   -    N.D. N.D.                                                                              N.D.                                                                              N.D.                            B cell                                                                        Daudl           -   -   -   -    -    -   -   -                               Raji            ++  ++  -   -    -    -   -   -                               RPMI1788        ++  ++  -   -    ± -   -   -                               RPMI8226        -   -   -   -    -    -   -   -                               Solid tumor                                                                   SK-MEL-37       -   -   -   -    -    -   ++  -                               SK-MEL-33       -   -   -   -    -    -   -   -                               Normal haemopoietic organ cells                                               Thymocyte       -   -   -   -    -    -   -   -                               Peripheral      -   -   -   -    -    -   -   -                               leukocyte                                                                     Monocyte        -   -   -   -    -    -   -   -                               Granulocyte     -   -   -   -    -    -   -   -                               Myeloid         -   -   -   -    -    -   -   -                               cell                                                                          __________________________________________________________________________     +++: >10.sup.7, ++: >10.sup.5, +: >10.sup.3, #+++: >10.sup.4, ±:           10.sup.2,                                                                     *positive cells: 1-5X                                                    

As a result, it has been found that Ta60a and Ta60b had the samereactivity Both of these antigens existed only in ATLV or HTLV-related(ATL-related) cells and did not exist in other T cells, and werenegative to RPMI8402, HPB-ALL, MOLT-3, Jurkat and IL-2-dependent NK3.3strain cells. However, they were positive to two cells among B cells inM-MHA, that is, Raji and RPMI1788. As regards the solid tumorcell-derived strains, they were negative to melanoma strains. shown inTable 1 (SK-MEL-37, SK-MEL-33) and to all of lung cancer, kidney cancer,stomach cancer and brain tumor strains. As regards normal blood cells,they were negative to all of thymocytes, peripheral leukocytes,monocytes, granulocytes and myeloid cells.

Ts60 existed in ATL-related cells (MT-2, HUT102 and HUT78) andexceptionally only in T cell-derived Jurkat cells, and did not exist inother cultured strain cells and normal cells. It is interesting to notein the results as to the intraplasm antigens by fluorescent antibodymethod that it was negative to Jurkat and positive to solid tumorSK-MEL-37, and was different from the cell membrane antigen in theexisting form.

ATV19a is an intraplasma antigen and existed in ATL-related cells, MT-2,MT-1, HUT102 and ATN-1, but was negative to all of the other cells.

EXAMPLE 7 Comparison of antibodies Ta60b and Ts60 in antibody titer tovarious cells (according to M-MHA method):

Antibody titers of TA60b, as compared on the basis of 50% positivevalue, were less positive in the order of MT-2, HUT102, MT-1, RPMI1788and Raji and were negative to HUT78 and Jurkat.

MT-2, HUT102: 10⁷⁻⁸

MT-1: 10⁷

RPMI1788, Raji: 10⁶

Ts-60 was positive to MT-2, HUT78, Jurkat and HUT102 and negative toMT-1, RPMI1788 and Raji.

MT-2: 10⁷⁻⁸

HUT78, Jurkat, HUT102: 10⁶

MT-1, RPMI1788, Raji: Negative

EXAMPLE 8 Reactivity of the monoclonal antibodies of the presentinvention to normal blood cells (according to M-MHA method):

As shown in Table 2, both Ta60a and Ta60b did not react at all withuntreated peripheral blood components, but when cultured in a mediumcontaining the following mitogens, both antigens turned positive.

(I) IL-2 10-25% (Biotest Co., West Germany)

(II) Phytohaemaglutinin (PHA) 20-50 μg/ml (GIBCO Co.)

(III) Concanavalin A (ConA) 20-25 μg/ml (GIBCO Co.)

On the other hand, Ts60 was negative under every conditions.

                  TABLE 2                                                         ______________________________________                                                           M-MHA inspection method                                               Number of                                                                             Number of positive cases                                   Cell group   test cases                                                                              Ta60a    Ta60b Ts60                                    ______________________________________                                        Peripheral leukocyte                                                          Fresh        12        0        0     0                                       Cultured control*                                                                          10        0        0     0                                       IL-2         7         6        6     0                                       PHA          5         5        5     0                                       ConA         5         5        5     0                                       Monocyte                                                                      Fresh        3         0        0     ND                                      Granulocyte                                                                   Fresh        2         0        0     0                                       Thymocyte                                                                     Fresh        6         0        0     0                                       Cultured control                                                                           3         0        0     0                                       Myeloid cell                                                                  Fresh        1         0        0     0                                       Spleen cell                                                                   Fresh        2         0        0     0                                       ______________________________________                                         *10% FCScontaining RPMI1640                                              

EXAMPLE 9 Reactivity of the monoclonal antibodies of the presentinvention to haemopoietic organ tumor cells (according to M-MHA method):

Results of inspection of 79 cases of haemopoietic organ tumors are shownin Table 3.

                  TABLE 3                                                         ______________________________________                                                          M-MHA inspection method                                             Number of Number of positive cases                                    Cell group                                                                              test cases  Ta60a    Ta60b Ts60                                     ______________________________________                                        ATL:                                                                          Fresh     16*.sup.1   10(5)    8(8)  0                                        Cultured* 14          9(5)     7(7)  0                                        T-CLL:                                                                        Fresh     1*.sup.2    1        0(1)  ND                                       T-ALL:                                                                        Fresh     7           0        0     0                                        T-ML:                                                                         Fresh     12          0        0(1)  0                                        Cultured  2           0        0     0                                        B-CLL:                                                                        Fresh     9           0(4)     0(3)  0                                        Cultured  5           0(2)     0(2)  0                                        B-ML:                                                                         Fresh     10          0(4)     3(1)  0                                        Null-ALL:                                                                     Fresh     11          1        1(1)  0                                        AML:                                                                          Fresh     4           2        2     0                                        AMOL:                                                                         Fresh     6           0        0     0                                        CML-BC:                                                                       Fresh     3           0(1)     1(1)  ND                                       ______________________________________                                         *10% FCScontainin RPMI1640                                               

Both of the antibodies Ta60a and Ta60b were positive to all the ATLcases, when weakly positive cases with antibody titers of less than 10⁵shown by the numbers in parentheses in Table 3 were included, and werenegative to all the other T cell-derived cases. They were positive toone case of T-CLL, which had already contained proviral DNA, and wasclinically diagnosed to be T-CLL, but could be regarded as ATL. Thus,both of these antigens are negative to T cell-derived malignanthaemopoietic organ diseases which are difficult to discriminate fromATL, and so these antibodies are useful for diagnosis. These antibodiesreacted with some cases of B cell-derived B-CLL and B-ML (malignantlymphoma of B cell) and also reacted, though weakly positively, withsome cases of myeloleukemia (AMC).

On the other hand, Ts60 was negative to all of the haemopoietic organdiseases.

EXAMPLE 10 Reactivity of the monoclonal antibodies of the presentinvention to IL-2 dependent cells

Cloned T cells 4E8 as IL-2 dependent cell strains [T cell strainsobtained by reacting peripheral blood from tuberculin positive healthyperson with PPD (a kind of tuberclosis cell membrane antigen) antigen ina test tube and culturing for a long time] were spread on a flat bottomculturing plate with 96 holes (made by Falcon Co.) at 2×10⁴ /0.2 ml ofculture liquor (10% FCS-containing RPMI1640 medium) for each well, andsubjected to reaction with antibodies at various concentrations at roomtemperature for 30 minutes.

Then, IL-2 was added thereto to make a final concentration of 5% (V/V)and the cells were cultured in the presence of 5% CO₂ - 95% O₂ at 37° C.for 72 hours Inhibition of IL-2 dependent cell propagation was observedby measuring the intake of 0.4 μCi of ³ H-methylthymidine for 4 hours

As a result, Ta60a showed an inhibition rate of 75%-95% up to aconcentration of 10⁻² -10⁻⁴ and Ta60b showed an inhibition rate of morethan 60% up to 10⁻² -10⁻⁴. On the other hand, Ts60 had no effect on theinhibition of cell propagation.

EXAMPLE 11

Reactivity of the monoclonal antibody of the present invention ATV19a tothe normal and malignant haemopoietic organ cells was investigatedaccording to M-MHA method, and the results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                        M-IF inspection method                                                          Number of Number of                                         Cell group        test cases                                                                              positive cases                                    ______________________________________                                        ATL:                                                                          Fresh             20        0                                                 Cultured control* 9         7                                                 IL-2              9         7                                                 T-CLL:                                                                        Fresh             1         0                                                 Cultured control  1         0                                                 IL-2              1         0                                                 T-cell lymphoma                                                               Fresh             5         0                                                 Cultured control  3         0                                                 IL-2              3         0                                                 B-cell lymphoma:                                                              Fresh             6         0                                                 Cultured control  3         0                                                 IL-2              2         0                                                 B-CLL:                                                                        Fresh             3         0                                                 ALL:                                                                          Fresh             7         0                                                 Cultured control  2         0                                                 IL-2              2         0                                                 AML:                                                                          Fresh             2         0                                                 AMOL:                                                                         Fresh             2         0                                                 CML-BC:                                                                       Fresh             1         0                                                 Infectious mononucleocyte                                                     Fresh             1         0                                                 Normal Peripheral                                                             leukocyte                                                                     Fresh             3         0                                                 Cultured control  10        0                                                 IL-2              10        0                                                 Monocyte                                                                      Fresh             2         0                                                 Granulocyte                                                                   Fresh             2         0                                                 Platelet                                                                      Fresh             2         0                                                 ______________________________________                                         *Culturing for 3 days                                                    

EXAMPLE 12 Reactivity of 9 antibodies detecting humanleukocyte-differentiating antigens to normal haemopoietic organ cells:

As shown in Table 5, antibodies Tpw40 and Tp120 reacted with 60-80% ofperipheral blood T cells, but did not react with B cells, monocytes,granulocytes, etc.

Antibody Tpw40 reacted with most of thymocytes, but Tp120 reacted onlywith 30-40% of thymocytes. Both antibodies detected panT cell antigen,but were different in specificity.

Four antibodies, Tsw32, TsA, TsB and Ts145 reacted with a fractioncontaining the peripheral blood T cells, but were unreactive to B cells,monocytes and granulocytes, and detected the so-called T cell subsetantigen. There was a differnce in reactivity to thymocytes among theantibodies, and Tsw22 reacted with 80-90% of thymocytes, whereasantibodies TsB and Ts145 reacted only with a very small portion ofcells.

Antibody Lp95 reacted with substantially all of the leukocyte groups andappears to detect pan leukocyte antigen.

Both antibodies Ls70 and LsA reacted with monocytes and other leukocytesand detected antigens existing in some cells of leukocytes.

                                      TABLE 5                                     __________________________________________________________________________    Reactivity of 9 antibodies detecting human leukocyte-differentiating          antigens                                                                      to normal haemopoietic organ cells                                                       Positive cell ratio by membrane fluorescent antibody method                   (%)                                                                           Number of                                                          Target cells                                                                             samples                                                                             Tpw40                                                                             Tp120                                                                             Tse32                                                                             TsA TsB Ts145                                                                             Lp95                                                                              Ls70                                                                              LsA                          __________________________________________________________________________    Thymocyte  4     90  30-40                                                                             80-90                                                                             20-40                                                                             0-20                                                                              0-10                                                                              60-80                                                                             0-10                                                                              0-10                         Peripheral blood                                                              T cell     10    60-70                                                                             70-80                                                                             25-35                                                                             50-70                                                                             40-60                                                                             10-30                                                                             60-80                                                                             10-20                                                                             20-30                        Non-T(B) cell                                                                            10    0-5 0-5 0-5 0-5 0-10                                                                              0-10                                                                              60-80                                                                             10-20                                                                             50-60                        Monocyte   4     0-5 0-5 0-5 0-5 0-5 0-5 80-90                                                                             90  50-60                        Granulocyte                                                                              4     0-5 0-5 0-5 0-5 0-5 0-5 90  20-30                                                                             0-5                          Spleen cell                                                                              3     50-60                                                                             50-60                                                                             40-50                                                                             30-40                                                                             10-20                                                                             10-20                                                                             60-70                                                                             20-30                                                                             40-50                        Activated lymphocyte                                                          PHA        8     70-90                                                                             30-40                                                                             40-70                                                                             50-70                                                                             50-70                                                                             10-30                                                                             70-80                                                                             40-60                                                                             10-30                        ConA       8     85-95                                                                             60-80                                                                             40-70                                                                             60-80                                                                             60-80                                                                             15-35                                                                             70-90                                                                             20-40                                                                             5-15                         Mixed cultured                                                                           8     60-70                                                                             60-70                                                                             20-30                                                                             50-70                                                                             30-50                                                                             20-40                                                                             70-80                                                                             20-40                                                                             10-20                        lymphocyte                                                                    Prolonged culturing                                                                      4     60-70                                                                             60-70                                                                             30-40                                                                             50-70                                                                             40-50                                                                             50-60                                                                             40-50                                                                             40-50                                                                             30-40                        of mixed cultured                                                             lymphocyte                                                                    __________________________________________________________________________

EXAMPLE 13 Reactivity of 9 antibodies detecting humanleukocyte-differentiating antigens to various haemopoietic organ tumors:

Reactivities to 60 cases of various haemopoietic organ tumors wereinvestigated and the results are shown in Table 6.

Antibody Lp95 detecting pan leukocyte antigen reacted with substantiallyall of the haemopoietic organ tumor cells. Antibodies Tpw40 and Tp120recognizing panT antigen were different in reactivity. Tpw40 reactedwith T-ALL and LL which were immature T cell lymphomas, and also reactedwith Ia antigen-negative Null-ALL. On the other hand, antibody Tp120reacted with T₂ lymphoma which was a mature T cell lymphoma, and ATL toa high degree. This antibody also reacted with B-CLL.

Both antibodies Tsw32 and TsA recognizing T subset antigen reacted withT-ALL and LL, and antibody TsA further had reactivities to T₂ lymphomaand ATL.

Antibody TsB recognizing T subset antigen reacted mainly with matured Tcell lymphomas such as T₂ lymphoma, ATL, etc. This antibody also reactedwith B-CLL. Antibody Ts145 did not react with any of the T celllymphomas to a high degree. Antibody Ls70 recognizing an antigenexisting in some of leukocytes reacted with matured T cell lymphoma andB-CLL.

Antibody LsA reacted with normal monocytes, and also with acutemonocytic leukocytes. This antibody did not react at all with T celllymphomas, B-CLL, etc.

Cell lines Ta60b and Ts145 were deposited on Dec. 2, 1988 in theFermentation Research Institute, Agency of Industrial Science andTechnology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305 Japanand were assigned accession numbes FERM BP-2170 and Ferm BP-2171respectively.

                                      TABLE 6                                     __________________________________________________________________________    Reactivity of 9 antibodies detecting human leukocyte-differentiating          antigens                                                                      to various haemopoietic organ tumors                                                         Number of positive cases by membrane fluorescent antibody                     method                                                                        Number of                                                      Haemopoietic organ tumors                                                                    test cases                                                                          Lp95                                                                             Tpw40                                                                             Tsw32                                                                             TsA                                                                              Tp120                                                                             TsB                                                                              Ls70                                                                             Ts145                                                                             LsA                          __________________________________________________________________________    Null type acute lymphatic                                                     leukemia (Null-ALL)                                                           Ia antigen-positive                                                                          6     2  1   0   0  0   0  0  1   0                            Ia antigen-negative                                                                          4     3  3   0   0  0   0  0  0   0                            T cell type acute lymphatic                                                                  7     7  7   5   5  3   2  0  1   0                            leukemia (T-ALL)                                                              Lymphoblast tumor (LL)                                                                       5     5  5   4   4  4   1  0  2   0                            Matured T cell lymphoma                                                                      8     8  3   3   8  6   8  7  2   0                            (T.sub.2 lymphoma)                                                            Adult T cell leukemia (ATL)                                                                  11    10 3   1   10 9   9  8  3   0                            B cell type chronic lymphatic                                                 leukemia (B-CLL)                                                                             8     6  0   0   5  8   8  8  3   0                            Acute myeloleukemia (AML)                                                                    6     4  1   0   0  0   0  2  0   2                            Acute monocytic leukemia                                                                     5     5  0   0   1  2   2  4  1   4                            (AMOL)                                                                        __________________________________________________________________________

We claim:
 1. Monoclonal antibody, Ta60b, detecting an antigen existingon human leukocytes which is non-reactive with thymocytes, produced byhybridoma cell line FERM BP-2170.
 2. Monoclonal antibody, Ts145,detecting an antigen existing on human leukocytes, produced by hybridomacell line FERM BP-2171.
 3. The hybridoma cell line which is Ta60b (FermBP-2170) or Ts145 (Ferm BP-2171).